J. Cosmet. Sci., 58, 505-517 (September/October 2007) The inhibitory effect of the components of Cornus officinalis on melanogenesis YASUHIKO NA WA, JUNKO ENDO, and TOMIHISA OHTA, Research & Development, Ichimaru Pharcos Co., Ltd., 318-1, Asagi, Motosu, Gifu 501-0475 (Y.N.), and Faculty of Pharmaceutical Sciences! Kanazawa University! Kakuma! Kanazawa, Ishikawa 920-0934 (Y.N., J.E.! T.O.)! ]apan. Accepted for publication May 22, 2007. Synopsis Five known compounds were isolated from a Cornus officinalis 50% ethanol extract (C. ojficinalis extract) and a hot water extract. We investigated the photochemical and pharmacological active compounds of C. officinalis hot water extract and ethanol extract. We understood that C. officinalis is a medicinal plant with potent free-radical-scavenging activity not only against reactive oxygen species (H202, superoxiside anion, hydroxyl radical, etc.) in a narrow sense, but also against many other free radicals (peroxynitrate, peroxyradi­ cal). It is estimated that the reduction effect with C. officinalis extract can block oxidative reaction on melanogenesis. Loganin and cornuside, the components in C. ojficinalis, showed a significant free-radical­ scavenging activity and inhibitory effects on melanogenesis. We report to prove the inhibitory effect of DVB-induced pigmentation in C. officinalis extract through its radical scavenging activity. INTRODUCTION Cornus officinalis Sieb. et Zucc. Cornaceae, which is commonly called Japanese cornel, is widely distributed in China and Korea. This plant produces light yellow flowers in early spring and bears cherry-like fruits in the autumn. The fruit, without the seed, is called sanshuyu, and has been used for at least 2,000 years in Chinese herbal medicine. It is used principally to reduce unusually active secretions, including copious sweating and excessive urine, and to "warm up and invigorate the yang of the loins." It is known that Cornus of/icinalis hot water extract has an antibacterial action, inhibiting the growth of Bacillus dysenteriae and Staphylococcus (1) and shows anti-histamine and anti­ acetylcholine activity in the small intestine of the guinea pig (2). Sanshuyu liqueur made from its fruit has been traditionally used as a nutritional drink. Further, its anti-diabetes effects and immunological enhancements have also been recognized by traditional medi­ cine. Identified as components in this natural medicine are iridoid-type monoterpene glycosides (3), morroniside (4), loganin, sweroside, tannins (5), and organic acids (tar­ taric acid, malic acid, and gallic acid). Tannins such as epigallocatechin and the deriva­ tives that are contained in green tea or tea are known to have an antioxidative effect and an astringent action, and are applied to cosmetic materials. Swertiamarin, an iridoid­ type monoterpene glycoside, is a bitter component in Swertia japonica, and is shown to 505

506 JOURNAL OF COSMETIC SCIENCE have a hair growth effect and is used in medicated cosmetic materials as a low skin primary irritation or a cumulative irritation component. Thus, sanshuyu or the compo­ nents have been applied as external agents (especially cosmetic) in the past. It is known that free radicals contribute to various vital reactions and induce many kinds of diseases (especially, in skin tissue, they induce dryness, aging, pigmentation, and cancer). Skin forms melanin in order to protect its tissues from UV rays, and excessive exposure to UV rays causes freckles. Pigmentation is influenced by melanogenesis through such agents as endothelin ( 6), histamine (7), pyrimidine dimer (8), and iNOS (9). We evaluated the inhibitory effect of DVB-induced pigmentation in C. officinalis extract through its radical-scavenging activity. Melanogenesis begins with the production of dopaquinone, a highly reactive o-quinone, and proceeds in three distinctive steps. The initial step is the production of cysteinyldopa by the rapid addition of cysteine to dopaquinone, which continues as long as cysteine is present. The second step is the oxidation of cysteinyldopa to give pheomelanin. The last step is the production of eumelanin. Ito (10) explained that the oxidative cascade from tyrosine to eu- or pheo­ melanin production is an important pathway of melanogenesis. We think that if the oxidative cascade were blocked to apply C. officinalis extract, it is possible to decrease melanogenesis and to develop this extract as a whitening agent. In this study, we have tried to isolate bioactive constituents with DPPH radical­ scavenging activity from hot water extract or ethanol extract that are well known pharmacological activities in C. officinalis. We evaluated phytochemically and pharma­ cologically bioactive constituents in C. officinalis and proved the inhibitory effect of DVB-induced pigmentation in C. officinalis extract through its radical-scavenging ac­ tivity. MATERIALS AND METHODS GENERAL PROCEDURES UV spectra were obtained on a Shimazu UV -1600 spectrophotometer. The NMR spectra were recorded on a J eol GSX-500 spectrometer. The MS were obtained on a J eol JMS SX-102 or JMS AX-5 MS spectrometer. HPLC was performed on a Japan Analytical Industry LC-908 system, equipped with normal-phase column (GKK Intersil PRER­ SIL, 20 x 250 mm) or a reversed-phase column (YMC-Pack R&D ODS, 20 x 250 mm). EGCG and ascorbic acid were purchased from Wako Pure Chemical Industries, Ltd. PLANT MATERIAL Sanshuyu (Cornus officinalis Sieb. et Zucc) was purchased from Matsuura Yakugyo Co., Ltd. and Uchida Wakanyaku Co., Ltd. EXTRACTION AND ISOLATION The dried fruits of sanshuyu (2.0 kg, cultivated in China) were extracted with hot water (8.0 kg, 100°C, 3 hr, using soxhlet). The hot-water extract (145 g) was evaporated to dryness. The dried extract (50 g) was subjected to HP-20 column chromatography (eluted with H20, 5% MeOH aq, MeOH, and EtOAc/MeOH= 1:1), and five fractions